CN105506053A - Detection method of strawberry pulp apoptosis - Google Patents
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Abstract
The invention relates to the field of apoptosis detection and particularly relates to a detection method of strawberry pulp apoptosis. The detection method comprises the following steps: cutting the pulp of a strawberry fruit into 1-2mm strips and performing enzymolysis treatment; filtering, centrifuging the filtrate and washing the obtained precipitate with a buffer solution to obtain a strawberry protoplast; adding a fluorescence probe into the strawberry protoplast and performing a warm bath for 3-5h at 35-38 DEG C; and observing the apoptosis condition under the ultraviolet exciting light of a fluorescence microscope. In the detection method, the strawberry protoplast is prepared first; through the warm bath of special time is performed on the strawberry protoplast and the fluorescence probe, the apoptosis condition of strawberry pulp can be observed under the fluorescence microscope, and the whole process is convenient and easy to implement. Moreover, the observation of the apoptosis condition of strawberries in different development stages helps understand the cell aging process of the strawberry pulp, lays a foundation for studying the fruit growth and development mechanism and is of important significance to the cultivation, transportation, storage and the like of commercial strawberries.
Description
Technical field
The present invention relates to apoptosis detection field, in particular to a kind of detection method of strawberry flesh cell apoptosis.
Background technology
Apoptosis (apoptosis) refers to as maintaining homeostasis, the orderly death autonomous by the cell of Gene Handling, is the earliest first to be proposed according to morphological feature by Kerr professor for 1972.Apoptosis is different from necrocytosis, and apoptosis is not a passive process, but active process, it relates to activation, the effect such as expression and regulation and control of series of genes.It is not under pathological conditions, from a kind of phenomenon of bulk damage, but for adapting to living environment better and a kind of death process initiatively striven for.During apoptosis, just as leaf or flower naturally wither and fall, this process is prevalent in animal and plant.
Apoptosis is one of focus of life science, detects apoptotic method and emerges in an endless stream.At present, for the method relative maturity that apoptosis in vitro detects, such as: flow cytometry, TUNEL detection method, DNA segment detection etc.For apoptosis in vivo detect reagent then studying among, various detection reagent constantly occurs.Wherein, AnnexinV, Synap-totagminI-C2A, ApoSense family molecule has certain advantage compared with other detection reagent.
Plant protoplast obtains " exposed cell " after cell removes cell walls.Since nineteen sixty Britain's plant physiologist Cocking adopts enzymolysis process to isolate great-hearted protoplastis from the tomato tip of a root first, have hundreds of plant so far with different tissues organ for material have successfully been obtained protoplastis.Protoplastis can efficiently take in the macromole such as foreign DNA, RNA, protein.By importing the reporter genes such as GUS, GFP in protoplastis, the researchs such as Assay of promoter activity, protein and organoid location can be carried out rapidly and accurately.
Strawberry is Rosaceae Fragaria perennial evergreen herbaceous plant, and its berry fragrance succulence, nutritious, deeply favor by producers and consumers, in the various berry in the world, its cultivated area and output occupy the 2nd.Therefore, research strawberry flesh cell apoptotic process, cultivate commodity strawberry, the aspects such as transport preservation are significant.
In view of this, special proposition the present invention.
Summary of the invention
The object of the present invention is to provide a kind of detection method of strawberry flesh cell apoptosis, strawberry process is obtained protoplastis by the method, specific fluorescent probe is selected to detect, by the apoptosis situation of the strawberry observation of cell to different development stage, significant to aspects such as the cultivation of commodity strawberry, transport, preservations.
In order to realize above-mentioned purpose of the present invention, spy by the following technical solutions:
A detection method for strawberry flesh cell apoptosis, comprises the following steps:
(a), get the pulp of strawberry fruit, carry out enzymolysis processing after being cut into 1-2mm slice, the mixed solution after enzymolysis filters, and obtains filtrate;
(b), by centrifugal for described filtrate, after the precipitation wash buffer obtained, obtain strawberry protoplast;
(c), in strawberry protoplast, add fluorescent probe, 35-38 DEG C of temperature bath 3-5h, is then placed in observation of cell apoptosis situation under fluorescent microscope uv excitation light;
The chemical structural formula of described fluorescent probe is as follows:
Apoptosis is a kind of spontaneous death process that organism normal cell occurs after being subject to physiology and pathological stimuli, is the process that an active, high-sequential, Gene Handling and a series of enzyme participate in.Apoptosis plays pivotal player in plant health survival processes, has vital role to the normal development of individuality.It has great significance in the maintenance of tissue differentiation, allelotaxis, body stable state.Body is while generation neonatal cell, and cell that is old and feeble and sudden change is eliminated by Apoptosis mechanism, and Organ and tissue is normally grown and metabolism.
The detection method of strawberry flesh cell apoptosis provided by the invention, carries out enzymolysis by after strawberry pulp slitting, and then centrifugal to filtering the filtrate obtained, centrifugal precipitation is rinsed and obtained strawberry protoplast; In strawberry protoplast, add specific fluorescent probe, the temperature through specified time is bathed, and be the apoptosis situation of observable strawberry pulp under fluorescent microscope, whole process is convenient and easy.In addition, by the apoptosis situation of the strawberry observation of cell to different development stage, understanding the process of strawberry flesh cell aging, laying the foundation for probing into fruit development mechanism, significant to aspects such as the cultivation of commodity strawberry, transport, preservations.
Preferably, strawberry pulp slice is immersed enzymolysis solution, then enzymolysis 50-60min on 23-27 DEG C of shaking table;
Wherein, described enzymolysis solution take water as solvent, its composition is as follows: cellulase 0.005-0.015g/ml, macerozyme 0.001-0.005g/ml, D-mannital 3.5-4.5mol/L, Repone K 0.01-0.03mol/L, calcium chloride 0.005-0.015mol/L, the volume percent that MES damping fluid accounts for described enzymolysis solution is the volume percent that 9%-12%, BSA account for described enzymolysis solution is 1.5%-2.5%.
By selecting specific enzymolysis solution, being beneficial to carry out sufficient enzymolysis to strawberry tissue, obtaining as far as possible many protoplastiss.
Preferably, in step (a), strawberry pulp slice first vacuumizes 25-30min after immersing enzymolysis solution, and vacuumizing power used is 2400-2500KPa.
First vacuumize process after first enzymolysis solution being immersed to strawberry pulp slice, be beneficial to allow tissue fully immerse enzyme liquid, vacuumize and select specific power, prevent from destroying strawberry cell.
Preferably, in step (a), described filtration adopts 580-600 object nylon wire to carry out;
The process of filtering is carried out on ice.
Nylon net filter removes tissue or the cell mass of non-thorough enzymolysis on the one hand, and the protoplast electrofusion obtained by enzymolysis on the one hand comes.In order to prevent protoplastis from sustaining damage, filtration procedure carries out on ice.
Preferably, in step (b), centrifugal rotating speed is 195-200g, and the centrifugal time is 100-150s.By of short duration centrifugal of low speed, with enrichment protoplastis, and reduce the infringement to protoplastis as far as possible.
Preferably, in step (b), described damping fluid take water as solvent, its composition is as follows: sodium-chlor 0.15-0.16mol/L, calcium chloride 0.12-0.13mol/L, the volume percent that Repone K 0.004-0.006mol/L, MES damping fluid accounts for described damping fluid is 0.5%-1.5%;
The number of times of described flushing is 2-3 time.
By selecting specific damping fluid to rinse the centrifugal protoplastis obtained, prevent from causing damage to protoplastis, after rinsing, protoplastis is less impure, and be easy to follow-up fluorescent dye, when carrying out fluorescence microscopy Microscopic observation, the visual field is cleaner.
Further, in step (c), the wavelength of fluorescent microscope is 349-352nm.This wavelength is observation of cell apoptosis situation under uv excitation light, and the cell under excitation light in blueness is apoptotic state, and greeny cell is then activated cell.
Test finds, the middle part pulp of strawberry prepares free protoplastis better effects if.Preferably, in step (a), the pulp of strawberry fruit takes from the middle part pulp of fruit.
Preferably, in step (c), the final concentration of described fluorescent probe in strawberry protoplast is 0.18-0.25 μm of ol/L.By adding the fluorescent probe of certain content, to make the cell of different activities better mark, observing effect is more directly perceived and accurate.
Further, also comprise between step (b) and step (c): strawberry protoplast is carried out viability examination;
Described viability examination adopts fluorescein diacetate to carry out.
First viability examination is carried out to obtained protoplastis, active to judge that the protoplastis obtained exists, to judge whether the operation entering next step.If detect and have vigor, then carry out fluorescent probe mark, the situation of observation of cell apoptosis; If detection debility, then need not carry out next step operation again.By this pre-judgement, save program.
Further, fluorescein diacetate is adopted to carry out viability examination concrete steps and be:
Get Protoplast suspension 200 ± 5 μ l, adding 10 ± 2 μ l mass concentrations is 0.01% diacetic acid fluorescein, and after leaving standstill 3-5min, drawing protoplastis mixed solution and drop on blood counting chamber, is 487-489nm basis of microscopic observation in wavelength.
Pass through viability examination, cell can only be observed and whether there is activity, apoptotic situation can not be observed, and adopt specific fluorescent probe to detect, according to the difference of fluorescence color, can distinguish and be in the apoptotic state of different developmental phases strawberry fruit.
Compared with prior art, beneficial effect of the present invention is:
(1) the present invention provides first and detects the apoptotic method of strawberry flesh cell, specific fluorescent probe is selected to detect apoptosis situation to obtained protoplastis, this method is easy and simple to handle, the process of strawberry flesh cell apoptosis can be observed intuitively, lay the foundation for probing into fruit development mechanism, significant to aspects such as the cultivation of commodity strawberry, transport, preservations;
(2) the present invention is also optimized the factor such as the combination of enzyme liquid, enzymolysis time, osmotic pressure regulator being separated strawberry pulp protoplastis, to obtain better protoplastis, provides basis for carrying out apoptosis detection;
(3) the present invention is before carrying out apoptosis, also carries out viability examination to strawberry protoplast, by this pre-judgement, saves program.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below.
Fig. 1 is that figure observed by the protoplastis microscope (40 times) in the embodiment of the present invention 1 obtained strawberry pulp green fruit period;
Fig. 2 is that figure observed by the protoplastis microscope (40 times) in the embodiment of the present invention 1 obtained strawberry gingko period;
Fig. 3 is that figure observed by the microscope (40 times) of the protoplastis viability examination in the embodiment of the present invention 1 obtained strawberry pulp green fruit period;
Fig. 4 is that figure observed by the microscope (40 times) of the protoplastis viability examination in the embodiment of the present invention 1 obtained strawberry gingko period;
Fig. 5 is that figure observed by the microscope (40 times) of strawberry pulp green fruit apoptosis in the period detection that the embodiment of the present invention 1 obtains;
Fig. 6 is that figure observed by the microscope (40 times) of strawberry gingko apoptosis in the period detection that the embodiment of the present invention 1 obtains;
Fig. 7 is that figure observed by the microscope (40 times) that the strawberry gingko apoptosis in period in another visual field that the embodiment of the present invention 1 obtains detects.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting the scope of the invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturers suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercially available acquisition.
Embodiment 1
The detection method of strawberry flesh cell apoptosis, comprises the following steps:
(1), enzymolysis solution and damping fluid is configured, enzymolysis solution take water as solvent, its composition is as follows: cellulase 0.01g/ml, macerozyme 0.002g/ml, D-mannital 4mol/L, Repone K 0.02mol/L, calcium chloride 0.01mol/L, the volume percent that MES damping fluid accounts for enzymolysis solution is the volume percent that 10%, BSA accounts for enzymolysis solution is 2%;
Damping fluid take water as solvent, and its composition is as follows: sodium-chlor 0.155mol/L, calcium chloride 0.125mol/L, and the volume percent that Repone K 0.005mol/L, MES damping fluid accounts for damping fluid is 1%;
(2) choose the strawberry fruit being in green fruit period and gingko etap in period, respectively, get middle part of fruit pulp respectively, cut 1-2mm slice, immerse enzymolysis solution respectively, then vacuumize 30min, vacuumizing power used is 2500KPa, fully immerses enzymolysis solution to allow organize;
(3), be then positioned over enzymolysis 60min on 25 DEG C of shaking tables, use tissue or the cell mass of the non-thorough enzymolysis of 600 object nylon net filter on ice, the centrifugal 120s of 200g;
(4), centrifugation uses wash buffer 2 times respectively, obtains the strawberry pulp Protoplast suspension in green fruit and gingko period respectively;
(5), each Protoplast suspension 200 ± 5 μ l is got, adding 10 ± 2 μ l mass concentrations is 0.01% diacetic acid fluorescein, after leaving standstill 5min, drawing protoplastis mixed solution drops on blood counting chamber, be observe under 40 power microscopes of 488nm in wavelength, bright field is observed as depicted in figs. 1 and 2, can see protoplastis sum; Dark-field is observed as shown in Figure 3 and Figure 4, and dark-field fluoresces is protoplastis; Can see that the protoplastis of different development stage strawberry exists according to Fig. 3 and Fig. 4 active;
(6), by fluorescent probe join respectively in the strawberry pulp Protoplast suspension in green fruit and gingko period, the final concentration of fluorescent probe in suspension is 0.2 μm of ol/L, 37 DEG C of temperature bath 4h;
(7), under fluorescent microscope 350nm uv excitation light (40 times), observation of cell apoptosis situation, if the cell under excitation light in blueness is apoptotic state, what take on a red color is just at the cell of apoptosis, greeny cell is then viable cell, and turn to blue cell theory daylight in the process just at apoptosis by green, thus observe apoptotic state.As shown in Figure 5, the apoptotic state of the strawberry pulp in gingko period as shown in Figure 6 and Figure 7 for the apoptotic state of the strawberry pulp in green fruit period.
As can be seen from Figure 5, the apoptotic cell number of the strawberry flesh cell in green fruit period is less, and as can be seen from Figures 6 and 7, the strawberry flesh cell in gingko period exists a large amount of just at the cell of apoptosis and apoptosis, can observe the apoptosis situation of cell intuitively.
Embodiment 2
The detection method of strawberry flesh cell apoptosis, comprises the following steps:
(1), enzymolysis solution and damping fluid is configured, enzymolysis solution take water as solvent, its composition is as follows: cellulase 0.005g/ml, macerozyme 0.001g/ml, D-mannital 3.5mol/L, Repone K 0.01mol/L, calcium chloride 0.005mol/L, the volume percent that MES damping fluid accounts for enzymolysis solution is the volume percent that 9%, BSA accounts for enzymolysis solution is 1.5%;
Damping fluid take water as solvent, and its composition is as follows: sodium-chlor 0.15mol/L, calcium chloride 0.12mol/L, and the volume percent that Repone K 0.004mol/L, MES damping fluid accounts for damping fluid is 0.5%;
(2) choose the strawberry fruit being in green fruit period and gingko etap in period, respectively, get middle part of fruit pulp respectively, cut 1-2mm slice, immerse enzymolysis solution respectively, then vacuumize 25min, vacuumizing power used is 2500KPa, fully immerses enzymolysis solution to allow organize;
(3), be then positioned over enzymolysis 55min on 27 DEG C of shaking tables, use tissue or the cell mass of the non-thorough enzymolysis of 580 object nylon net filter on ice, the centrifugal 100s of 195g;
(4), centrifugation uses wash buffer 3 times respectively, obtains the strawberry pulp Protoplast suspension in green fruit and gingko period respectively;
(5), each Protoplast suspension 200 ± 5 μ l is got, adding 10 ± 2 μ l mass concentrations is 0.01% diacetic acid fluorescein, after leaving standstill 3min, drawing protoplastis mixed solution drops on blood counting chamber, be observe under 40 power microscopes of 487nm in wavelength, it is active that dark-field can see that the protoplastis of different development stage strawberry exists;
(6), by fluorescent probe join respectively in the strawberry pulp Protoplast suspension in green fruit and gingko period, the final concentration of fluorescent probe in suspension is 0.18 μm of ol/L, 38 DEG C of temperature bath 3h;
(7), under fluorescent microscope 349nm uv excitation light, observation of cell apoptosis situation, if the cell under excitation light in blueness is apoptotic state, what take on a red color is just at the cell of apoptosis, greeny cell is then viable cell, and turn to blue cell theory daylight in the process just at apoptosis by green, thus observe apoptotic state.The apoptotic state of the strawberry pulp in green fruit period is with shown in Fig. 5; Shown in same Fig. 6-7 of apoptotic state of the strawberry pulp in gingko period.The apoptosis situation of the strawberry flesh cell in green fruit period and gingko period intuitively can be arrived by microscopic examination.
Embodiment 3
The detection method of strawberry flesh cell apoptosis, comprises the following steps:
(1), enzymolysis solution and damping fluid is configured, enzymolysis solution take water as solvent, its composition is as follows: cellulase 0.015g/ml, macerozyme 0.005g/ml, D-mannital 4.5mol/L, Repone K 0.03mol/L, calcium chloride 0.015mol/L, the volume percent that MES damping fluid accounts for enzymolysis solution is the volume percent that 12%, BSA accounts for enzymolysis solution is 2.5%;
Damping fluid take water as solvent, and its composition is as follows: sodium-chlor 0.16mol/L, calcium chloride 0.13mol/L, and the volume percent that Repone K 0.006mol/L, MES damping fluid accounts for damping fluid is 1.5%;
(2) choose the strawberry fruit being in green fruit period and gingko etap in period, respectively, get middle part of fruit pulp respectively, cut 1-2mm slice, immerse enzymolysis solution respectively, then vacuumize 30min, vacuumizing power used is 2400KPa, fully immerses enzymolysis solution to allow organize;
(3), be then positioned over enzymolysis 50min on 23 DEG C of shaking tables, use tissue or the cell mass of the non-thorough enzymolysis of 600 object nylon net filter on ice, the centrifugal 150s of 200g;
(4), centrifugation uses wash buffer 2 times respectively, obtains the strawberry pulp Protoplast suspension in green fruit and gingko period respectively;
(5), each Protoplast suspension 200 ± 5 μ l is got, adding 10 ± 2 μ l mass concentrations is 0.01% diacetic acid fluorescein, after leaving standstill 5min, drawing protoplastis mixed solution drops on blood counting chamber, be observe under 40 power microscopes of 489nm in wavelength, it is active that dark-field can see that the protoplastis of different development stage strawberry exists;
(6), by fluorescent probe join respectively in the strawberry pulp Protoplast suspension in green fruit and gingko period, the final concentration of fluorescent probe in suspension is 0.25 μm of ol/L, 35 DEG C of temperature bath 5h;
(7), under fluorescent microscope 352nm uv excitation light, observation of cell apoptosis situation, if the cell under excitation light in blueness is apoptotic state, what take on a red color is just at the cell of apoptosis, greeny cell is then viable cell, and turn to blue cell theory daylight in the process just at apoptosis by green, thus observe apoptotic state.Same Fig. 5 of apoptotic state of the strawberry pulp in green fruit period; Shown in same Fig. 6-7 of apoptotic state of the strawberry pulp in gingko period.The apoptosis situation of the strawberry flesh cell in green fruit period and gingko period intuitively can be arrived by microscopic examination.
The chemical structural formula of fluorescent probe used in embodiment 1-3 is as follows:
Although illustrate and describe the present invention with specific embodiment, however it will be appreciated that can to make when not deviating from the spirit and scope of the present invention many other change and amendment.Therefore, this means to comprise all such changes and modifications belonged in the scope of the invention in the following claims.
Claims (10)
1. a detection method for strawberry flesh cell apoptosis, is characterized in that, comprises the following steps:
(a), get the pulp of strawberry fruit, carry out enzymolysis processing after being cut into 1-2mm slice, the mixed solution after enzymolysis filters, and obtains filtrate;
(b), by centrifugal for described filtrate, after the precipitation wash buffer obtained, obtain strawberry protoplast;
(c), in strawberry protoplast, add fluorescent probe, 35-38 DEG C of temperature bath 3-5h, is then placed in observation of cell apoptosis situation under fluorescent microscope uv excitation light;
The chemical structural formula of described fluorescent probe is as follows:
2. the detection method of strawberry flesh cell apoptosis according to claim 1, is characterized in that, in step (a), described enzymolysis processing is:
Strawberry pulp slice is immersed enzymolysis solution, then enzymolysis 50-60min on 23-27 DEG C of shaking table;
Wherein, described enzymolysis solution take water as solvent, its composition is as follows: cellulase 0.005-0.015g/ml, macerozyme 0.001-0.005g/ml, D-mannital 3.5-4.5mol/L, Repone K 0.01-0.03mol/L, calcium chloride 0.005-0.015mol/L, the volume percent that MES damping fluid accounts for described enzymolysis solution is the volume percent that 9%-12%, BSA account for described enzymolysis solution is 1.5%-2.5%.
3. the detection method of strawberry flesh cell apoptosis according to claim 2, is characterized in that, in step (a), described filtration adopts 580-600 object nylon wire to carry out;
The process of filtering is carried out on ice.
4. the detection method of strawberry flesh cell apoptosis according to claim 3, is characterized in that, in step (a), strawberry pulp slice first vacuumizes 25-30min after immersing enzymolysis solution, and vacuumizing power used is 2400-2500KPa;
Preferably, in step (b), centrifugal rotating speed is 195-200g, and the centrifugal time is 100-150s.
5. the detection method of strawberry flesh cell apoptosis according to claim 1, it is characterized in that, in step (b), described damping fluid take water as solvent, its composition is as follows: sodium-chlor 0.15-0.16mol/L, calcium chloride 0.12-0.13mol/L, the volume percent that Repone K 0.004-0.006mol/L, MES damping fluid accounts for described damping fluid is 0.5%-1.5%;
The number of times of described flushing is 2-3 time.
6. the detection method of strawberry flesh cell apoptosis according to claim 1, is characterized in that, in step (c), the wavelength of fluorescent microscope is 349-352nm.
7. the detection method of strawberry flesh cell apoptosis according to claim 1, is characterized in that, in step (a), the pulp of strawberry fruit takes from the middle part pulp of fruit.
8. the detection method of strawberry flesh cell apoptosis according to claim 1, is characterized in that, in step (c), the final concentration of described fluorescent probe in strawberry protoplast is 0.18-0.25 μm of ol/L.
9. the detection method of the strawberry flesh cell apoptosis according to any one of claim 1-8, is characterized in that, also comprise between step (b) and step (c): strawberry protoplast is carried out viability examination;
Described viability examination adopts fluorescein diacetate to carry out.
10. the detection method of strawberry flesh cell apoptosis according to claim 9, is characterized in that, adopts fluorescein diacetate to carry out viability examination concrete steps to be:
Get Protoplast suspension 200 ± 5 μ l, adding 10 ± 2 μ l mass concentrations is 0.01% diacetic acid fluorescein, and after leaving standstill 3-5min, drawing protoplastis mixed solution and drop on blood counting chamber, is 487-489nm basis of microscopic observation in wavelength.
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| CN115521897A (en) * | 2022-09-22 | 2022-12-27 | 鲁东大学 | Method for extracting protoplast from forest strawberry callus |
| CN116183470A (en) * | 2022-12-22 | 2023-05-30 | 中国林业科学研究院林业研究所 | Woody plant cell programmed death detection method based on flow sorting |
| CN116183470B (en) * | 2022-12-22 | 2024-04-05 | 中国林业科学研究院林业研究所 | Woody plant cell programmed death detection method based on flow sorting |
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